Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck. Survival for patients with oral squamous cell carcinoma (OSCC) has not improved in the past half century, primarily because of the high level of metastatic disease at the time of diagnosis and the lack of effective therapies for those metastases. The goal of this project is to understand molecular pathways by which cystatins (Cys) assist OSCC cells in evading immunesurveillance. We recently compared the mRNA profiles of patient- matched primary and metastatic OSCC cell lines via DNA microarrays, observing that the combined expression levels of Cys (A, B, C & M) were markedly higher in metastatic cells than in their parental primary cells. However, the levels CB, their targeted proteinase, remained unchanged. CB exerts two distinct functions: (1). It is an effector proteinase of TNF-related Apoptosis Inducing Ligand (TRAIL)-mediated tumor cell apoptosis; TRAIL is crucial for the antimetastatic effect of NK cells; (2) CB surface expression protects CTL and NK cells from activation-induced perforin-mediated self lysis. Hypothesis: Overexpressed Cys confer OSCC a metastatic phenotype, probably via two mechanisms: 1) Cys enhance resistance to TRAIL- induced apoptosis mediated by NK cells and 2) Cys reduce the self-protection mechanism of antitumor immune effector cells leading to an increased rate of apoptosis among these cells. Aim 1. Determine effects of overexpression and RNAi-based gene silencing of cystatins A and C in TRAIL- and NK cell-mediated killing of murine SCC cells in vitro. Aim 2. Investigate how stable overexpression and RNAi-mediated silencing of cystatins A and C in murine SCC cells affects their growth, metastasis and interaction with host immune system in vivo. Our studies will focus on induced overexpression and siRNA mediated silencing of Cys in murine SCC cells modifying their susceptibility to TRAIL- and NK cell-mediated killing. Next, we will explore how stable overexpression or silencing of CysA and -C in these cells affects their growth, metastasis and interaction with host immune system, using an orthotopic and immunocompetent murine OSCC model. These studies are aimed at determining how Cys produced by tumor cells corrupt host antitumor immunity and allow metastatic progression. Insights gained from these studies will be used to design therapeutic strategies for restoring immune competence of OSCC patients to prevent and eradicate OSCC metastasis. [unreadable] [unreadable] [unreadable]